Processes for the production of amidase

ABSTRACT

An amidase or nitrilase is made by continuous culture under carbon limitation using a carbon source which includes, respectively, either (a) an amide or amide precursor or (b) a nitrile or nitrile precursor. Novel enzymes have particular stability. A novel microorganism is Rhodococcus rhodochrous NCIMB 40756 and is capable of producing a particularly stable amidase. The novel amidase, and the amidase made by the defined process, are effective for converting (meth)acrylamide to ammonium (meth)acrylate, for instance in or after the polymerisation of the acrylamide.

This invention relates to the production of enzymes by fermentationprocesses and the use of the enzymes as catalysts. It also relates tonovel microorganisms particularly suitable for the production of certainimproved enzymes.

It is known to convert amide molecules to their corresponding acid oracid salt. This is desired in particular in the case of acrylamide.

Conversion of (meth) acrylamide to its acid or salt is required on anindustrial scale for production of (meth) acrylic acid monomer or a saltthereof.

Conversion of (meth) acrylamide to (meth) acrylic acid monomer is alsodesired for the purification of (meth) acrylamide homopolymer orcopolymer of (meth) acrylamide with other monomers. Hydrolysis ofresidual (meth) acrylamide monomer is desirable as a way of meetingenvironmental concerns about the monomer.

It is known to use enzyme catalysts to hydrolyse residual monomeric(meth) acrylamide for the purification of (meth) acrylamide-containingpolymers. Processes of this type are described in EP-A-0329324,EP-A-0329325 and WO92/05205.

It has also been suggested by Hawaz et al, in Applied and EnvironmentalMicrobiology, September 1994, p.3343-3348, that it would be desirable touse enzyme catalysts for the large scale production of acrylic acid ifappropriate enzymes were available. In this paper Nawaz et al describe aRhodococcus species which can use acrylamide as a growth substrate andproduce an amidase enzyme. The bacterial cells were grown in batchculture.

It is generally found that when enzyme catalysts are used in thehydrolysis of monomeric (meth) acrylamide that the presence of the(meth) acrylamide monomer has a detrimental effect on the long termstability of the enzyme. There is a tendency for the enzyme catalyst tolose activity over time. This necessitates addition of further enzymecatalyst to return the catalytic activity to its original level. Thelower the stability of the enzyme catalyst the less economical theprocess.

It is also known to convert nitrile molecules to their correspondingacid or acid salt. This can be carried out by the direct conversion ofnitrile to acid using a nitrilase enzyme as catalyst. As used herein, a"nitrilase" is an enzyme which carries out the direct conversion of anitrile to its corresponding acid without release of an amideintermediate. Nitrilases have been described in, inter alia,EP-A-444,640, JP-B-632596, Biotechnol. Appl. Biochem. 15, 283-302(1992), Appl. Microbiol. Biotechnol. (1990) 34:322-324, Biotechnol.Appl. Biochem. 11, 581-601 (1989) and J. Bacteriol., September 1990,pages 4807 to 4815.

As with amidases, these enzymes often show a tendency to destabilise inthe presence of large amounts of substrate (nitrile). As with amidases,instability results in a less economic process.

The normal way of cultivating microorganisms which produce an amidase ornitrilase is batch culturing. In this method an initial amount of themicroorganism is placed in a growth medium containing a large excess ofall nutrients necessary for growth of the microorganism. Growth proceedseither until it is terminated by harvesting the batch of culturedmicroorganism or until termination of growth due to exhaustion ofnutrients or toxification of the growth medium due to build-up ofby-products from the microorganism.

An alternative culture method is known as continuous culture. Continuousculture methods are generally known. In the most commonly used methodsthe culture, containing microorganism and growth medium and usually inliquid form, is continuously removed from the culture vessel and isreplaced at the same rate with fresh growth medium so that the volume ofliquid culture remains constant. Under steady state conditions thereplication rate of the microorganism is governed by addition of newgrowth medium such that the biomass concentration of the culture isconstant.

In continuous culture methods it is usual to provide one element whichis metabolised to depletion in the culture medium and governs the steadystate biomass concentration. This is known as the growth limitingelement. All other elements are generally in excess.

EP-A-393916 describes a method of culturing a microorganism continuouslyunder nitrogen limitation, which results in the microorganisms producedcontaining elevated levels of an amidase enzyme.

In GB-A-1379728 continuous culture appears to be conducted underconditions of carbon limitation, the carbon source being material suchas glycerol, lactate or other material which can lead to Kreb's cycleintermediates. Preferably only one such carbon source is present in theculture medium.

It would be desirable to be able to produce an amidase or nitrilaseenzyme improved in stability over the generally used enzymes. It wouldalso be desirable to be able to produce an amidase or nitrilase enzymehaving greater catalytic activity. In particular, it would be desirableto provide an enzyme which is particularly suitable for use as theenzyme in processes such as those described in WO92/05205 andEP-A-0329324 and EP-A-0329325.

According to a first aspect of the invention there is provided a processof producing an amidase or nitrilase enzyme comprising

cultivating in continuous culture a microorganism capable of producingthe enzyme in a growth medium comprising a carbon source

wherein the culture is conducted under carbon limitation and wherein ifan amidase enzyme is being produced the carbon source includes an amideor amide precursor and if a nitrilase enzyme is being produced thecarbon source includes a nitrile or nitrile precursor.

It will be appreciated that in this process the microorganism ispreferably an inducible microorganism, in contrast to the constitutivemicroorganism used in GB-A-1379728.

The invention reduces catabolite repression by limiting the amount ofmetabolite available (as a result of the carbon limitation). Cataboliterepression has previously been proposed in some processes to increaseyield but we surprisingly find that the process of the invention has anumber of other advantages. The microorganisms produced by this processexhibit an enzyme activity per gram cell weight which is much improvedover activity obtained in equivalent microorganisms produced by batchculturing methods.

A particularly unexpected advantage is the improvement in stability ofthe enzyme produced by the microorganisms cultivated according to theprocess of the invention, in comparison with enzyme produced byequivalent microorganisms obtained by batch culturing methods. In anacrylamide or acrylonitrile reactor the enzyme can show a decrease inactivity over time which is greatly reduced in comparison with batchcultured enzymes of the same type.

The process can surprisingly yield enzyme of particular value for use inprocesses such as those described in EP-A-0329324 and 0329325 and,especially, WO92/05205.

In this invention when we say "continuous culture" we include the trulycontinuous processes commonly used. We also include any process ofcultivating microorganisms which is not a true batch process. Thus weinclude semi-continuous processes. In a true batch process excess of allnutrients is present initially and growth is allowed to continue untilit becomes self-limiting. In the invention the nutrient or nutrientswhich provide the growth-limiting element must be added continuously orsemi-continuously to the culture vessel throughout growth, so that thegrowth-limiting element is never present in excess. A continuous processof the invention often involves continuous removal of microorganism asit grows. However, this is not essential. The process may be carried outfor instance with excess of all elements except carbon being presentinitially and carbon being introduced gradually in order to limit rateof growth, which continues until the other nutrients are exhausted. Suchprocesses are sometimes referred to as "fed batch" processes. In theinvention truly continuous processes are preferred.

The process of the invention may be used to produce an amidase ornitrilase enzyme. Generally only one of these enzymes is produced in asingle process, although it is possible to produce mixtures of enzyme.This can be done for instance by culturing a mixture of compatiblemicroorganisms. Preferably the process of the invention is used toproduce an amidase enzyme.

In the invention it is essential, where an amidase is produced, that theculture is conducted under carbon limitation. Other elements, forinstance nitrogen, oxygen and phosphorus, are in excess. The exactproportions of these elements which are used in the growth medium willbe determined by the composition of the particular microorganism beingcultured and can be planned by establishing the relative amounts ofthese and other elements in the microorganism.

In the invention it is also essential, where an amidase is produced,that the source of carbon includes an amide or amide precursor. An amideprecursor is a compound which can be metabolised to an amide by themicroorganism being cultured or is otherwise converted to an amide inthe growth medium.

Suitable amides include acrylamide, methacrylamide, acetamide andpropionamide and N-substituted derivatives of these. Mixtures may beused. Acetamide is preferred.

Suitable amide precursors include nitriles such as acrylonitrile,methacrylonitrile and acetonitrile and mixtures thereof. Mixtures ofamides and amide precursors may be used.

For production of amidase, the amide or amide precursor generally formsat least 20 mol %, preferably at least 30 mol % and more preferably atleast 50 mol % of the total carbon source. Often the amide or amideprecursor forms at least 80 mol % of the carbon source. Preferably thecarbon source consists substantially only of amide and/or amideprecursor.

It is particularly preferred, for production of amidase, for the carbonsource to consist substantially only of acetamide or a precursorthereof.

In the invention it is essential, where a nitrilase is produced, thatthe source of carbon includes a nitrile or nitrile precursor. A nitrileprecursor is a compound which can be metabolised to a nitrile by themicroorganism being cultured or is otherwise converted to a nitrile inthe growth medium.

Suitable nitriles include acrylonitrile, methacrylonitrile, acetonitrileand propionitrile. Mixtures may be used.

For production of nitrilase, the nitrile or nitrile precursor generallyforms at least 20 mol %, preferably at least 30 mol % and morepreferably at least 50 mole % of the total carbon source. Often thenitrile or nitrile precursor forms at least 80 mole % of the carbonsource. Preferably the carbon source consists substantially only ofnitrile and/or nitrile precursor.

Where the carbon source comprises an amide or mixture of amides thiswill generally contribute towards the nitrogen and oxygen components ofthe growth medium. Generally it will form only part of the oxygensource. The amide may form only part of the nitrogen component or it mayprovide the whole of the nitrogen component of the growth medium.Whether this is possible will depend on the composition of themicroorganism being cultured. For instance, if the amide has anitrogen:carbon ratio greater than that of the microorganism, it may bepossible to provide the amide in amounts which result in a growth mediumwhich is carbon-limited but contains excess nitrogen, both elementsbeing provided solely by the amide. If the nitrogen:carbon ratio in theamide is lower than that of the microorganism being cultured it willgenerally be necessary to provide a further source of nitrogen inaddition to the amide in order for nitrogen to be present in excess.

Similar considerations apply to nitriles as components of the carbonsource. These can also contribute towards the nitrogen component of thegrowth medium.

The carbon source may also include other carbon-containing compounds inaddition to the amide, amide precursor, nitrile or nitrile precursor.Suitable compounds are compatible with and capable of being metabolisedby the microorganism and compatible with other components of the growthmedium. Suitable compounds include carboxylic acids and derivativesthereof, alcohols, sugars, starches and other carbohydrates.

The growth medium should contain sources of all other necessaryelements. These may include nitrogen, oxygen, phosphorus, sulphur,hydrogen, potassium, sodium, calcium, magnesium, copper, zinc,manganese, iron and other metals, chlorine and vitamins.

These materials may be provided in the form of any compound which can bemetabolised by the microorganism. Suitable components of the growthmedium include organic acids, for instance carboxylic acids and theiralkali metal or ammonium salts; chlorides, sulphates and other salts ofsodium, and the other metals listed above; acids of phosphorus and theirsalts; vitamins; salts of trace metals; amino acids.

The culture is conducted under conditions of pH and temperature suitablefor the microorganism being cultured. The pH of the growth medium isgenerally between 6 and 8, preferably above 6.5. The pH may be below7.5, and is often substantially neutral. The temperature which is mostsuitable for the culturing process will depend on the microorganismselected. Generally temperature is from 20 to 42° C., often 25 to 37° C.

If the microorganism chosen produces the required enzyme or enzymesconstitutively then no special conditions are required to ensure theenzyme is produced. Certain microorganisms produce enzymes inducibly andtherefore require special conditions, for instance particularconstituents of the growth medium, in order to induce them to producethe required enzyme. Induction of enzyme production can be carried outin any known manner. For instance, nitrilase may be induced as describedin Biotechnol. Appl. Biochem. 15, 283-302 (1992), then Biotechnol.Biochem. 11, 581-601 (1989) or Appl. Microbiol. Biotechnol. (1990)34:322-324 and EP-A-444,640.

The method of the invention may be used to produce any amidase ornitrilase enzyme. Known microorganisms which are suitable for producingan amidase enzyme include bacterial sources, for instance strains of thegenera Brevibacterium, Pseudomonas, Alcaligenes, Arthrobacter,Corynebacterium, Mycobacterium, Lactobacillus, Micrococcus, Nocardia,Strentomyces, Rhodococcus, Microbacterium, Bacteridium and mixedcultures of Brevibacterium and Bacillus. Suitable yeast and fungiinclude strains of Fusarium and Aspergillus. Suitable bacterial sourcesfor nitrilase include strains of the genera Corynebacterium, Nocardia,Bacillus, Bacteridium, Micrococcus and Brevibacterium, for instanceRhodococcus rhodochrous J1, Rhodococcus ATCC 39484, Rhodococcusrhodochrous K22, Fusarium oxysporum and Pseudomonas chloraraphis.

A particularly preferred microorganism is the novel microorganism alsoprovided by the invention. This is a microorganism which is Rhodococcusrhodochrous strain NCIMB 40756 or mutant thereof having the ability toproduce an amidase. A sample of this strain was deposited at theNational Collections of Industrial and Marine Bacteria Limited (NCIMB),23 St. Machar Drive, Aberdeen AB2 1RY, Scotland, UK on Jul. 14, 1995under the Budapest Treaty and has the accession number NCIMB 40756. Allrestrictions imposed by the depositor on the availability to the publicof the deposited biological material will be irrevocably removed uponthe granting of the patent.

The above strain showed the following results on analysis:

The cell wall diamino acid is meso DAP. Mycolic acids are present. Thefatty acid profile shows the following acids in the indicatedpercentages:

    ______________________________________                                        tetradecanoic          14.6%                                                    hexadecenoic 19.0%                                                            hexadecanoic 19.4%                                                            octadecenoic 23.5%                                                            tuberculostearic (10-methyloctadecanoic)  25.2%.                            ______________________________________                                    

Biochemical testing gave the following results:

    ______________________________________                                                        10 days, 30° C.                                        ______________________________________                                        Decomposition of:                                                               Adenine +                                                                     Tryrosine +                                                                   Urea -                                                                        Decomposition of:                                                             Inositol.sup.1 -                                                              Maltose.sup.1 +                                                               Mannitol.sup.1 +                                                              Rhamnose.sup.1 -                                                              Sorbitol.sup.1 +                                                              m-hydroxybenzoic acid.sup.2 (+)                                               Sodium adipate.sup.2 -                                                        Sodium benzoate.sup.2 +                                                       Sodium citrate.sup.2 +                                                        Sodium lactate.sup.2 +                                                        Sodium glutamate.sup.2 -                                                      L-tyrosine.sup.2 +                                                            Glycerol.sup.1 +                                                              Trehalose.sup.1 -                                                             p-hydroxybenzoic acid.sup.2 +                                                 D-mannose.sup.1 (+)                                                           Acetamide.sup.2 +                                                             D-galactose.sup.1 -                                                           Enzymatic tests:                                                              α-glucosidase +                                                         Cysteine arylamidase -                                                        Valine arylamidase -                                                          Growth in the presence of:                                                    5% NaCl +                                                                     Sodium azide.sup.3 -                                                        ______________________________________                                         .sup.1 1% w/v                                                                 .sup.2 0.1% w/v                                                               .sup.3 0.02% w/v                                                              (+) weak positive                                                        

This strain is capable of producing amidase enzyme.

A further preferred microorganism is Rhodococcus rhodochrous strainNCIMB 40757 or a mutant thereof having the ability to produce anitrilase. A sample of this strain was deposited at the NationalCollections of Industrial and Marine Bacterial Limited (NCIMB), 23 St.Machar Drive, Aberdeen AB2 1RY, Scotland, UK on Aug. 8, 1995 inaccordance with the provisions of the Budapest Treaty and has theaccession number NCIMB 40757.

The strain deposited under NCIMB 40757 showed the following results onanalysis:

The cell wall diamino acid is meso DAP. The fatty acid profile shows thefollowing acids in the indicated percentages:

    ______________________________________                                        tetradecanoic  2.1%                                                             pentadecanoic 2.8%                                                            hexadecenoic 24.7%                                                            hexadecanoic 25.9%                                                            heptadecenoic 6.2%                                                            heptadecanoic 3.1%                                                            octadecenoic 25.0%                                                            octadecanoic 1.9%                                                             tuberculostearic  7.0%.                                                     ______________________________________                                    

Biochemical testing gave the following results:

    ______________________________________                                        Decomposition of:                                                               Adenine -                                                                     Tyrosine +                                                                    Urea -                                                                        Growth in Presence of:                                                        5% NaCl +                                                                     Dextrose azide (+)                                                            Growth on sole carbon sources:                                                Inositol (+)                                                                  Maltose +                                                                     Mannitol +                                                                    Rhamnose -                                                                    Sorbitol +                                                                    m-hydroxybenzoic acid +                                                       Sodium adipate +                                                              Sodium benzoate +                                                             Sodium citrate +                                                              Sodium lactate +                                                              Sodium glutamate -                                                            L-tyrosine +                                                                  Glycerol +                                                                    Trehalose +                                                                   p-hydroxybenzoic acid +                                                       D-mannose +                                                                   Acetamide +                                                                   D-galactose (+)                                                               Enzyme Tests:                                                                 Rosco discs. 4 hours. 37° C.                                           α-glucosidase -                                                         Cysteine arylamidase -                                                        Valine arylamidase -                                                        ______________________________________                                         (+) weak positive                                                        

This strain is capable of producing nitrilase enzyme and is described infurther detail in our copending Application No. 9525372.0.

Yields and specific activities, and also other characteristics of themicroorganism cultivated in the method of the invention, may be improvedby selection procedures, eg after mutagenesis.

Amidase enzymes produced by the process of the invention may be used forthe conversion of amides to their corresponding acids or salts, Thus asecond aspect of the invention also comprises a method of converting anamide to the corresponding acid or acid salt comprising

producing an amidase enzyme by cultivating in continuous culture amicroorganism capable of producing an amidase enzyme in a growth mediumcomprising a carbon source which includes an amide or amide precursorwherein the culture is conducted under carbon limitation

and introducing the enzyme into an environment containing the amide.

This method is particularly useful for the conversion of unsaturatedamides to their corresponding unsaturated acids or acid salts. Suitableamides include acrylamide and methacrylamide, which are converted intoammonium (meth) acrylate, which can then be further reacted to form(meth) acrylic acid or other salts if desired.

The method of the second aspect of the invention may be used for thepurification of polymers which have been formed from acrylamide monomer,with or without comonomer (polyacrylamides). This purification may becarried out in any conventional manner, for instance as described inU.S. Pat. No. 4,687,807 or in EP-A-0329,324 and EP-A-0329,325. Thus theenzyme may be maintained in contact with the polymer in particulate formin a reverse phase emulsion (which may be dehydrated) or it may becontacted with polymer gel which may then be dried. The enzyme has goodactivity when the emulsion or gel is maintained in contact with theenzyme at elevated temperatures, e.g. 40 to 95° C., especially 50 to 80°C.

The concentration of acrylamide monomer in a polyacrylamide environmentmay be as high as 2,000 ppm and is generally from 100 to 1,000 ppm,often 200 to 1,000 ppm. The enzyme may be used to effect a reduction inthe acrylamide monomer content of the polyacrylamide. Where the originalacrylamide monomer content is relatively high, for instance around 2,000ppm, the method of the invention can effect a reduction to 500 ppm orbelow. Where the concentration of acrylamide monomer is lower, forinstance up to 1,000 ppm, monomer levels can be reduced to below 200 ppmor even as low as 50 ppm or 10 ppm or less.

In a preferred process of the invention, we make a polymer of (meth)acrylamide by a process which comprises providing an aqueouspolymerisable mixture containing (meth) acrylamide in a reaction vessel,exothermically polymerising the polymerisable mixture and recovering theresultant polymer from the reaction vessel, and in this process theresidual (meth) acrylamide content of the polymer is reduced byincorporating in the polymerisable mixture the amidase made by theprocess of the invention.

The method of the second aspect of the invention may also be used forindustrial scale production of ammonium acrylate by hydrolysis ofacrylamide. The ammonium acrylate may be converted to a different saltor to the acid form or may be used directly as a monomer inpolymerisation processes.

The acrylamide conversion method of the invention can be a continuousprocess. In such a process the concentration of ammonium acrylate in thereactor and thus in the reactor output is preferably from 5 to 25%, morepreferably not more than 20%, most preferably 10 to 15%. Highconcentration acrylamide (for instance a 300 to 400 g/l solution) is fedinto the reactor to maintain a concentration of acrylamide of forinstance 3 to 45 g/l, often 20 to 30 g/l.

Preferably however the acrylamide conversion method of the invention isa fed batch process. In such a process high concentration acrylamide(for instance a 300 to 400 g/l solution) is fed into a reactorcontaining the amidase enzyme and the concentration of ammonium acrylateis allowed to rise to a specified value, for instance 5 to 25%,preferably at least 10%, often 15 to 20%, and the reaction is thenstopped. During the reaction the acrylamide concentration is preferablymaintained at from 3 to 45 g/l, preferably at least 15 g/l, generallyaround 27 to 30 g/l.

The amidase enzyme may be introduced into the reaction mixture(containing acrylamide monomer or impure polyacrylamide) in any suitableform. It may for instance be introduced in the pure form, having beenextracted from the cultured microorganism before use as a catalyst. Theextraction method used should ensure that the activity and stability ofthe enzyme are not lost.

It may also be introduced in a semi-pure form, for instance as liquidculture or a bacterial cell fraction such as intact cells or crushedcells. The amidase may be introduced as a crude, impure enzyme solution.It may be supported or immobilised on a carrier. Suitable carriersinclude cross-linked polymeric matrix, for instance cross-linkedpolyvinyl alcohol or cross-linked polyacrylamide. The enzyme may beincorporated into a carrier in any suitable form, for instance as pure,extracted enzyme or as intact bacterial cells. Preferably the amidaseenzyme is introduced in the form of intact bacterial cells or supportedin a cross-linked polymeric matrix.

The amide conversion reaction is generally carried out at a temperatureof from 0 to 50° C., often 4 to 30° C.

We find that in this amidase conversion method the enzyme producedaccording to the process of the invention gives far greater stabilitythan the batch-produced enzymes previously used. In particular thehalf-life (period taken for enzymic activity to drop to half itsoriginal level) of the amidase enzyme used in the method of theinvention can be increased two-, four- and often tenfold in comparisonwith equivalent amidase enzymes produced using the same microorganismsby batch methods.

Nitrilase enzyme produced by the process of the invention may be usedfor the conversion of nitriles to their corresponding acid (or salt).

A third aspect of the invention comprises a method of converting anitrile to its corresponding acid or acid salt comprising

producing a nitrilase enzyme by cultivating in continuous culture amicroorganism capable of producing a nitrilase enzyme in a growth mediumcomprising a carbon source which includes a nitrile or nitrile precursorwherein the culture is conducted under carbon limitation

and introducing the enzyme into an environment containing the nitrile.

This method is particularly useful for conversion of unsaturatednitrites. Suitable nitriles include acrylonitrile and methacrylonitrile,which are converted into ammonium (meth) acrylate.

The method of the third aspect of the invention may be used for thepurification of polymers which have been formed from (meth)acrylonitrile monomer, with or without comonomer. This purification maybe carried out in any known manner.

The method of the third aspect of the invention may also be used forindustrial scale production of ammonium acrylate. Conditions should bechosen to allow optimum activity of the enzyme and may be conventional.

Enzyme may be introduced into the reaction mixture in any of the formsdescribed above for amidase. The nitrile conversions are generallycarried out at temperatures of from 0 to 50° C., often 4 to 30° C.

Nitrile conversion processes in which the nitrilase can be used aredescribed in our co-pending applications having numbers 9525372.0 and9525374.6.

The nitrilase enzyme produced according to the process of the inventionagain has stability to the reaction mixture greater than batch-producedequivalent enzymes.

The enzyme product of the continuous culture process of the invention isa novel product in itself. Accordingly a fourth aspect of the inventionalso provides an amidase or nitrilase enzyme obtainable by a processcomprising cultivating in continuous culture a microorganism capable ofproducing the enzyme in a growth medium comprising a carbon sourcewherein the culture is conducted under carbon limitation and wherein ifan amidase enzyme is being produced the carbon source includes an amideor amide precursor and if a nitrilase enzyme is being produced thecarbon source includes a nitrile or nitrile precursor.

The product is obtainable by any of the processes discussed above inconjunction with the process of the first aspect of the invention.Preferably the product is an amidase enzyme.

It is not clear as yet exactly why the enzyme product of the inventiongives its improved results. It is possible that in microorganismscapable of producing more than one form of amidase or nitrilase enzymethe conditions of the continuous culturing process of the inventioninduce production of a more stable form in preference to a less stableform. This stability may be due to higher molecular weight or to adifferent amino acid sequence. It is also possible that the enzymeproduced according to the invention has a quaternary structure differentfrom equivalent batch-produced enzymes; for instance it may be in theform of an aggregate of two or more protein sub-units.

The product of the invention is preferably an amidase and obtainable bya process as described above in which the source of carbon issubstantially only acetamide and in which the microorganism isRhodococcus rhodochrous strain NCIMB 40756 or a mutant thereof able toproduce an amidase, or one whose yield and other characteristics havebeen improved by selection procedures, eg after mutagenesis.

The enzyme product of the invention may be prepared from microorganismsbut it may also be prepared by cloning, using conventional techniques.

The preferred amidase product of the invention exhibits excellentstability in the presence of amides which it is required to hydrolyse.In particular, the invention provides an amidase enzyme which has ahalf-life t1/2 of 80 to 300 days, preferably 126 to 200 days, at 5° C.in an aqueous environment at pH 7 in a sequential fed batch process inwhich the acrylamide concentration is 27 g/l to 30 g/l and the ammoniumacrylate concentration rises from 0 to 150 g/l in each batch. In thistest, "days" are days of reaction and do not include dormant daysbetween batches.

The invention also includes the use of the novel enzymes and/ormicroorganisms that will produce them for converting (meth) acrylamideto ammonium (meth) acrylate either in bulk or in a polymer ofacrylamide, e.g. as described in EP-A-0329324 and EP-0A-0329325. Inparticular the invention includes making a polymer of (meth) acrylamideby a process which comprises providing an aqueous polymerisable mixturecontaining (meth) acrylamide in a reaction vessel, exothermicallypolymerising the polymerisable mixture and recovering the resultantpolymer from the reaction vessel, and in this process the residual(meth) acrylamide content of the polymer is reduced by incorporating inthe polymerisable mixture the novel enzyme and/or microorganism.

Generally the polymerisation proceeds exothermally to a temperature thatin commercial practice is nearly always well above 50° C., typicallyabove about 55 or 60° C. and often above about 70° C., e.g. up to 80 or90° C. Generally the entire temperature rise is due to the exotherm andthe process of the invention is preferably conducted on a polymerisablemixture that has a concentration such that there will be an exothermicrise of at least 20° C. and often at least 30° C. and frequently atleast 40° C., e.g. to a temperature of 80 or even 90° C.

Thus in the invention the amidase is incorporated in the polymerisablemixture, generally before any polymerisation occurs, and so is exposedto the presence of a large amount of monomer and to the significantexotherm, and it would have been thought that these two conditions wouldhave been undesirable. However we have surprisingly found that theenzyme of the invention is effective under these conditions.

The enzyme and/or microorganism may be used in the manner and in theprocesses described in EP-A-0329324, EP-A-0329325 and WO92/05205.

The entire disclosure of each of those published Applications of AlliedColloids Limited and David Farrar is hereby incorporated by reference.

The product of the invention may alternatively be a nitrilase. Thisenzyme is also highly stable in the presence of reaction mixture.

The invention will now be illustrated with reference to the followingexamples.

EXAMPLE 1

The original isolate Rhodococcus rhodochrous strain NCIMB 40756 grown inbatch culture and containing amidase enzyme or in which amidase enzymecan be induced is transferred to continuous culture. The culture medium,which is shown in the table below, is designed to grow Rhodococcusrhodochrous strain NCIMB 40756 continuously under carbon limitation,where acetamide is the sole carbon and nitrogen source.

    ______________________________________                                        Component     Amount present/liter                                            ______________________________________                                        K.sub.2 HPO.sub.4                                                                             7 g                                                             KH.sub.2 PO.sub.4   3 g                                                       Acetamide 1.1 g                                                               NaCl 0.1 g                                                                    CaCl.sub.2.6H.sub.2 O 0.2 g                                                   MgSO.sub.4.7H.sub.2 O 0.5 g                                                   Vitamins 0.1 ml                                                               Trace Metals   1 ml                                                         ______________________________________                                    

Under steady-state culture at a dilution rate of 0.04 h⁻¹, (achievedduring the period from 160 to 390 hours), elevated levels of specificamidase activity were observed, as shown in Table 1 below.

                  TABLE 1                                                         ______________________________________                                                      Specific Amidase                                                  Culture time Activity Biomass                                                 (hrs) (U/mg) (g/l)                                                          ______________________________________                                         0            not measured                                                                              0.10                                                   50 not measured 0.10                                                         100 11 0.09                                                                   150 22 0.52                                                                   200  9 0.31                                                                   250 12 0.34                                                                   300 16 0.37                                                                   350 15 0.34                                                                   390 15 0.31                                                                 ______________________________________                                    

COMPARATIVE EXAMPLE 1

The original isolate Rhodococcus rhodochrous strain NCIMB 40756 grown inbatch culture and containing amidase enzyme or in which amidase enzymecan be induced is transferred to batch culture. The initial compositionof the batch culture medium is shown in the table below.

    ______________________________________                                        Component     Amount Present/liter                                            ______________________________________                                        K.sub.2 HPO.sub.4                                                                           7 g                                                               KH.sub.2 PO.sub.4 3 g                                                         Acetamide 2 g                                                                 Sodium Acetate 10 g                                                           CaCl.sub.2.6H.sub.2 O 0.1 g                                                   MgSO.sub.4.7H.sub.2 O 0.5 g                                                   Vitamins 0.1 ml                                                               Trace Metals 1 ml                                                           ______________________________________                                    

During batch culture comparatively low specific amidase activity wasobserved. Results are shown in Table 2 below.

                  TABLE 2                                                         ______________________________________                                                      Specific Amidase                                                  Culture time Activity Biomass                                                 (hrs) (U/mg) (g/l)                                                          ______________________________________                                        0             not measured                                                                              0.10                                                  2 not measured 0.10                                                           4 not measured 0.15                                                           6 not measured 0.20                                                           7 2.6 0.25                                                                    8 2.4 0.30                                                                    10  2.3 0.75                                                                  12  2.6 1.70                                                                ______________________________________                                    

EXAMPLE 2

The cells of the Rhodococcus rhodochrous strain grown as described inExample 1 are immobilised in cross-linked polyacrylamide beads asfollows:

a paste consisting of cells separated from the culture medium issuspended in chilled buffer and added to a mixture of monomer andcross-linker also in chilled buffer. The water-soluble component of aredox initiator system is added immediately afterwards. Thecell/monomer/initiator mixture is then transferred to a stirred resinpot containing mineral oil and chilled surfactant and the second redoxinitiator component, soluble in both liquid phases, is added to initiatepolymerisation. Upon polymerisation the cells are entrapped in thecross-linked polymer beads.

The amidase-active immobilised cells are transferred to a reactor andsuspended in water at 5° C. and acrylamide added to give a concentrationof 30 g/l. The amidase in the immobilised cells catalyses the hydrolysisof the acrylamide to produce ammonium acrylate. When the reactoracrylamide concentration is reduced to 27 g/l, sufficient acrylamidesolution (at a concentration of 355 g/l), is automatically added to thereactor to raise the acrylamide concentration to 30 g/l. This automaticfeeding procedure continues until the reactor ammonium acrylateconcentration has risen to 150 g/l. Upon completion of a batch, whichtakes around 18 hrs, the imnobilised Rhodococcus rhodochrous strainNCIMB 40756 cells are separated from the suspending monomer solution,resuspended in water and acrylamide added in a repetition of theprocedure described above. The specific amidase activity is determinedfrom the time taken for a known quantity of cells to complete a batchand the specific yield is determined from the amount of ammoniumacrylate produced during a series of batches, as follows: ##EQU1##Results are shown in Table 3 below. The half life of the amidase was 126days.

    ______________________________________                                                     Specific Yield                                                                            Specific Amidase                                       Reactor (g ammonium Activity                                                  Operation Time acrylate per g (μmoles/min/g                                (days) dry weight cells) dry weight cells)                                  ______________________________________                                         2            75         255                                                     4 130 254                                                                     6 200 252                                                                     8 260 250                                                                    10 320 248                                                                    12 390 247                                                                    14 450 245                                                                    20 610 230                                                                    22 660 225                                                                  ______________________________________                                    

COMPARATIVE EXAMPLE 2

Cells of the Rhodococcus rhodochrous strain grown as described inComparative Example 1 are immobilised in cross-linked polyacrylamidebeads in the same way as described in Example 2. The amidase-activeimmobilised cells are transferred to a reactor and suspended in water at5° C. and acrylamide added to give a concentration of 30 g/l. Theremaining procedure is as described in Example 2. Results are shown inTable 4 below.

    ______________________________________                                                     Specific Yield                                                                            Specific Amidase                                       Reactor (g ammonium Activity                                                  Operation Time acrylate per g (μmoles/min/g                                (days) dry weight cells) dry weight cells)                                  ______________________________________                                        2            20          83                                                     4 35 67                                                                       6 48 52                                                                       8 57 42                                                                       10  66 38                                                                     12  73 35                                                                   ______________________________________                                    

The results above show the improved stability and overall productivityof the amidase enzymes produced by the process according to the presentinvention. The activity of the amidase of the invention was consistentlyconsiderably greater than that of the amidase produced by batch culture.Furthermore, the amidase of the invention retained a far greaterproportion of its activity over time. For instance the amidase of theinvention underwent a drop of 8 U/g in activity in the period from 2 to12 days, i.e. 3.1%. The amidase produced by the batch culturedmicroorganism underwent a drop of 48 U/g in the same period, that is56.5%. The half-life of the enzyme was 8 days.

What is claimed is:
 1. A process for producing amidase or nitrilaseenzyme comprisinga) cultivating a microorganism capable of producing theenzyme in continuous culture in a growth medium comprising a carbonsource wherein the culture is conducted under carbon limitation andwherein when an amidase enzyme is being produced the carbon sourceincludes an amide or amide precursor and when a nitrilase enzyme isbeing produced the carbon source includes a nitrile or nitrileprecursor, wherein the microorganism is Rhodococcus rhodochrous strainNCIMB 40756 or a mutant thereof having the ability to produce anamidase; and b) recovering the resulting enzyme.
 2. A process accordingto claim 1 in which the carbon source consists of one or more amides ornitrites.
 3. A process according to claim 1 in which the enzyme is anamidase.
 4. A process according to claim 3 in which an amidase isproduced and the carbon source is substantially only acetamide.
 5. Amethod of converting an amide to the corresponding acid or acid saltcomprisinga) introducing an amidase enzyme into an environmentcontaining an amide wherein the amidase(1) has been produced by aprocess comprising cultivating a microorganism capable of producing theenzyme in continuous culture in a growth medium comprising a carbonsource wherein the culture is conducted under carbon limitation andwherein the carbon source includes an amide or amide precursor or (2) isan amidase having a half-life t1/2 of 80 to 300 days in an aqueousenvironment at pH7 in a sequential fed batch process in which theacrylamide concentration is 27 g/l to 30 g/l and the ammonium acrylateconcentration rises from 0 to 150 g/l in each batch or (3) is introducedby means of a microorganism which is Rhodococcus rhodochrous strainNCIMB 40756 and b) recovering the corresponding acid or acid salt ofsaid amide.
 6. A method according to claim 5 in which the amide isacrylamide and is converted to ammonium acrylate.
 7. A method accordingto claim 5 in which the concentration of amide in the amide-containingenvironment is at least 15 g/l.
 8. A method according to claim 5 inwhich the amide is residual acrylamide monomer contaminating apolyacrylamide or copolymer of acrylamide with other monomer or monomersand the amidase or microorganism is contacted with the polymer at atemperature of 40 to 95° C.
 9. A method according to claim 5 comprisingincorporating the enzyme or microorganism in an aqueous polymerisablemixture comprising (meth)acrylamide in a reaction vessel, exothermicallypolymerising the mixture and recovering the resultant polymer from thereaction vessel, whereby the inclusion of the enzyme or microorganismproduces a resultant polymer that contains less (meth)acrylamide than aresultant polymer wherein no enzyme or microorganism has been included.